Liposomes vesicles whose binding envelope consists of bi- or multilayer of lipid molecules have been long recognised as drug delivery systems which can improve therapeutic and diagnostic effectiveness of many drugs and contrast agents. Experiments with a number of different antibiotics and X-ray contrast agents have shown that better therapeutic activity or better contrast with a higher level of safety may be achieved by encapsulating drugs and contrast agents with liposomes. Great interest in liposomes as encapsulating systems for drugs has revealed that a successful development and commercialisation of such products requires reproducible methods of large scale production of lipid vesicles with suitable characteristics. Consequently, a search for methods which will consistently produce liposome vesicles of the required size and concentration, size distribution and entrapping capacity regardless of the nature of lipid mixture have been initiated. Such methods ought to provide liposomes with consistent active substance to lipid ratio while respecting currently accepted good manufacturing practices. As a result of the search, and due to the fact that the liposome behaviour can vary substantially with various production parameters, many different methods of manufacture have been proposed so far.
Conventional liposome preparation methods include a number of steps in which multi- or the bilayer-forming components (phospholipids or mixtures of phospholipids with other lipids e.g. cholesterol) are dissolved in a volatile organic solvent or solvent mixture in a round bottom flask followed by evaporation of the solvent under conditions (temperature and pressure) which will prevent phase separation. Upon solvent removal, a dry lipid mixture, usually in form of a film deposit on the walls of the reactor, is hydrated with an aqueous medium which may contain dissolved buffers, salts, conditioning agents and an active substance to be entrapped. Liposomes will form in the hydration step whereby a proportion of the aqueous medium becomes encapsulated in the liposomes. The hydration can be performed with or without energising the solution by means of stirring, sonication or microfluidisation with subsequent extrusion through one or more polycarbonate filters. The free non-encapsulated active substance can be separated for recovery and the product is filtered, sterilised, optionally lyophilised, and packed.
Hydration, more than any other step, influences the type of liposomes formed (size, number of lipid layers, entrapped volume). The nature of the dried lipid, its surface area, and its porosity are of particular importance. Thus it has been established that the hydration and entrapping process are most efficient when the film of dry lipids is kept thin. This means that greater the lipid quantity, greater the surface for deposition of the lipids is required, it also means that even though glass beads and other inert insoluble particles are used to increase the surface area available for film deposition, the thin film method remains largely a laboratory method.
Other methods of making liposomes involving injection of an organic solutions of lipids into an aqueous medium with continuous removal of solvent, use of spray drying, lyophilization, microemulsification and microfluidization, etc. have been proposed a number of publications or patents such as for example U.S. Pat. No. 4,529,561, U.S. Pat. No. 4,572,425, etc.
An attempt to solve problems of the scale-up of liposome production has been described in the U.S. Pat. No. 4,935,171 (Vestar). There is disclosed a method for preparing liposomes in commercial quantities by forming a homogeneous and uniform lipid film in a thin-film evaporator through evaporation of the organic solvent. After drying of the thin lipid film which is formed on the inner wall of the evaporator, the deposit is in situ hydrated with an aqueous phase under agitation provided by the rotor. Although the solution proposed in this document seems to be a step in the right direction the lipid film surface to the reactor volume ratio is only slightly, if not marginally, better than that of the round-bottom flasks used on laboratory scale. The reactor's space time yield or productivity is still far too low for the process to be economically sound and competitive.
Different aspects of the liposome manufacturing have been addressed and a number of improvements and different solutions to the problem of scale-up have been proposed. Documents such as for example WO-A-86/00238, WO-A-87/00043, U.S. Pat. No. 4,737,323, U.S. Pat. No. 4,753,788, and U.S. Pat. No. 4,781,871 have suggested use of rapid freezing of previously prepared multi lamellar vesicles with subsequent freeze and thaw treatment to improve their entrapment capacity, use of extrusion technique of multilamellar liposomes to improve their size distribution, etc.
So far there has been no suggestion towards a large scale industrial method whose control of production parameters will allow reproducible process in which large volumes of liquid will be processed within a relatively small reactor space. All known processes of pilot or industrial scale would, typically, be linked to small batches in which processing of large volumes of dilute liposome solutions would require a lot of floor and reactor space as well as handling of large volumes of solutions and solvents. In reality due to relatively low space time yields or productivity of reactors these methods are too cumbersome and far too costly for a large scale commercial production.